Methods for labeling nucleotides, labeled nucleotides and useful intermediates

ABSTRACT

The present invention provides a labeled nucleotide having formula:  
                 
 
     where X is an aliphatic diamine, and the labeling moiety comprises a label and a spacer, where the spacer is coupled at one end to a platinum atom and at the other end to the label, which spacer comprises a chain having at least four atoms. The invention further provides a method for labeling a nucleotide, kits for labeling a nucleotide, and kits containing labeled nucleotides.

[0001] The invention relates to methods for labeling nucleotides usinglinkers (linking moieties between labels and bio-organic molecules,which linkers are based on platinum compounds).

[0002] Platinum (coordination) compounds have been consideredinteresting molecules for a very long time. For a review of thesecompounds and their uses we refer to Reedijk et al. (Structure andBonding 67, p.53-89, 1987). Especially Cis-platinum has received a lotof attention as a possible anti-tumour drug. This anti-tumour reactivityof platinum compounds originates from their having at least two reactivegroups (preferably cis-oriented towards each other), which make itpossible to cross-link DNA molecules, thereby inhibiting the replicationof these DNA molecules.

[0003] The British patent application 2 148 891 discloses cis-platinumcomplexes, which are six-coordinated. The platinum is attached to twohalogens or hydroxy groups, two additional halogens and to an ethylenediamine derived group, such as 1,2-diamino-2-methylpropane or1,2-diamino-2-methylbutane. The complexes are said to have an excellentanti-tumor effect.

[0004] In the European patent application four-coordinated complexes ofplatinum to 2,3-alkyl-1,4-butanediamine and two halogens are describedfor their anti-tumor effect.

[0005] Different four-coordinated platinum complexes are described inthe European patent application 0 386 243. The complexes comprise adiamine bidentate ligand and two 2-arylalkanoic acid or3-aryl-2-oxoalkanoic acid ligands. These complexes are stated to have astrong growth inhibiting action on certain leukemia cells and are usedfor their oncostatic activity.

[0006] U.S. Pat. No. 4,207,416 discloses ethylenediamine-platinum(II)2,4-dioxopyrimidine complexes as having a high anti-tumor activity andlow mammalian toxicity.

[0007] A different use of platinum (coordination) compounds has beendisclosed in PCT application (WO92/01699) wherein a platinum compoundhaving only two reactive moieties (denominated as leaving groupstherein) is reacted with a fluorescein to obtain a labeled platinumcompound which can bind (non-covalently) to a nucleic acid, preferablyat the N-7 position of a guanine residue.

[0008] Several methods for labeling nucleotides have been described inthe literature. For a long time, the standard method has been to useradioactive isotope labeling. However, there are a number of problemsassociated with the use of radioisotopes, such as potential healthhazards, disposal problems and instability problems.

[0009] In order to overcome these problems, Dale et al., Biochemistry,14, (1975), 2447-2457, have proposed to use direct covalent mercurationas a labeling technique for nucleotides and polynucleotides. It wasfound, that cytosine and uracil may be mercurated at their C5-positionunder mild conditions. Further, Gebeyehu et al., Nucleic Acids Research,15, (1987), 4513-4534, have reported that adenine and cytosine may belabeled with biotin derivatives through an aliphatic linker of from 3 to17 atoms.

[0010] A major drawback of these known methods is that they are notsuitable for labeling all different nucleotides. For instance, Dale etal. reported that their covalent mercuration method leads to negativeresults for adenine, thymine and guanine bases. In some cases, forexample when only a few residues of a certain nucleotide are present ina certain nucleic acid or when the terminating nucleotide residue of anucleic acid has to be labeled, it is desired to have at one's disposala method for labeling any nucleotide residue.

[0011] The present invention provides such a method. The method forlabeling nucleotides of the invention comprises the steps of:

[0012] reacting a reactive moiety of a linker of the formula

[0013] wherein X represents any stabilizing bridge and wherein A and Brepresent the same or different reactive moieties, with an electrondonating moiety of a spacer, which spacer comprises a chain having atleast four atoms and at least one heteroatom in the chain, which spacerfurther comprises said electron donating moiety at one end of the chainand a reactive moiety at the other end of the chain;

[0014] reacting the reactive moiety of said spacer with a label;

[0015] reacting the other reactive moiety of said linker with anucleotide.

[0016] According to the invention, the linker may first be attached tothe nucleotide and then to the spacer, or vice versa and the spacer mayfirst be coupled to the label and then to the linker or vice versa.

[0017] The reactive moiety of the spacer may be any reactive moiety thatwill enable the reaction between the spacer and the label in such amanner that a labeling moiety comrpising a label and a spacer is formed,which labeling moiety is sufficiently stable.

[0018] The main purpose for labeling nucleotides is that these labelednucleotides can be incorporated in nucleic acid molecules. Modifiednucleotides, especially those wherein a (bulky) label is attached to thenucleotide, are often built-in into nucleic acids with a much lowerefficiency. The methods according to the invention result in labelednucleotides which are built-in into nucleic acids with a higherefficiency than the labeled nucleotides available to date. This isprobably for due to the selection of the spacers according to theinvention in combination with the platinum-based linkers according tothe invention.

[0019] The label to be used according to the invention is not critical.In principle all labels which can be attached to a nucleotide and areemployed to date can be used. These labels may be radioactive labels,enzymes (which need reaction with a substrate to be detected), specificbinding pairs components such as avidin, streptavidin or biotin,biocytin, iminobiotin, colloidal dye substances, fluorochromes(rhodamin, etc.), reducing substances (eosin, erythrosin, etc.),(coloured) latex sols, digoxigenin, metals (ruthenium), metal sols orother particulate sols (selenium, carbon and the like), dansyl lysin,Infra Red Dyes, coumarines (amino methyl coumarine), antibodies, proteinA, protein G, etc. The invention has most benefits with bulkier labelssuch as biotin, avidin, streptavidin, digoxygenin or a functionalequivalent thereof.

[0020] The invention is not limited to nucleotides or nucleosides assuch; derivatives and functional equivalents are also included. Theusual nucleotides adenine, thymidine, cytosine, guanine and uridine arepreferred. Especially the purines are preferred which have a very goodincorporation rate.

[0021] For coupling of the spacer to the platinum linker an electrondonating moiety is required. In a preferred method the electron donatingmoiety is an amine or a thiolate anion, which have both proven to bevery succesful. It was found that aromatic amines, such as imidazoles orpurines, are capable of forming very strong bonds to platinum and thusare very suitable for use as the electron donating moiety.

[0022] The spacer is an important aspect of the present invention; itprovides the easiest coupling between label and linker. For avoidingsteric hindrance in incorporation of the nucleotide into the nucleicacid it should at least be four atoms long, preferably it is at leastfour carbon atoms long and has at least one heteroatom in that carbonchain. A heteroatom confers a certain amount of rigidity on the spacer.This rigidity provides an additional assurance that steric factors willnot obstruct a convenient linking of a nucleotide and a label. It ispreferred that at least one heteroatom is an oxygen atom, whichpositively effects the hydrophilicity of the spacer.

[0023] Preferably, the spacer comprises no more than 20 carbon atoms inthe chain, which is preferably an essentially non-branched chain, thuscausing no steric hindrance. The reason for this will be clear.

[0024] A highly preferred spacer is 1,8-diamino-3,6-dioxaoctane, hereinreferred to as Dadoo. Dadoo is a very flexible compound with a distalprimary amine group and a size that makes it very suitable for use asspacer according to the invention.

[0025] Another highly preferred spacer of the invention is anoligolysine or a polylysine. Due to their structure and conformation,these molecules create the most convenient environment for an optimalinteraction among the actual label, the nucelotide and the platinum. Anadditional advantage of the use of lysine chains as the spacer is, thatby altering the number of lysine units in the chain, the optimalconditions for specific labels and nucleotides or nucleic acids can beattained. Given a certain application, the skilled person will easilydetermine how many lysine units are required for optimum results.

[0026] An especially interesting labeling moiety comprising a label anda spacer, has the formula

[0027] or the formula

[0028] wherein X represents any stabilizing bridge, Z and Z′ represent anon-leaving ligand and n is an integer of from 2 to 10.

[0029] Accordingly, the linker-spacer-label-system, or labelingsubstance, with the labeling moiety of formula (II) or formula (III) hasthe formula

[0030] or the formula

[0031] wherein A, X, Z, Z′ and n have the above meanings.

[0032] The non-leaving ligands Z and/or Z′ are preferably an NH₃, NH₂R,NHR₂ or NR₃ group, wherein R represents an alkyl group having from 1 to6 carbon atoms, because these ligands have an even smaller leaving-groupcharacter than other non-leaving ligands.

[0033] The interesting feature of using the labeling moieties havingformulas (II) and (III) is that both the nucleotide and the actual labelhave the benefit of being bonded directly to a platinum atom, while atthe same time these moieties are sufficiently far apart to avoid sterichindrance.

[0034] The linkers according to the invention preferably are platinumcompounds wherein X represents an aliphatic diamine. In a preferredembodiment of the invention, one or both of the nitrogen atoms of thealiphatic diamine are shielded. A suitable manner of shielding thesenitrogen atoms consists of substitution with one or two alkyl groups offrom 1 to 6 carbon atoms, preferably methyl groups. This is advantageousin that hydrogen bonding between the triphosphate group of a nucleotideand the stabilizing bridge is prevented. Preferably, a diamine having2-6 carbon atoms is use, most preferably an ethylene diamine group,which 's well-known for its stabilizing effect on this class of platinumcompounds. In this case, the linker has the formula

[0035] wherein G represents hydrogen or an alkyl group of from 1 to 6carbon atoms and A and B represent the same or different reactivemoieties.

[0036] The coupling or reactive moieties A and B are preferably the sameand selected from the group consisting of NO₃ ⁻, SO₃ ⁻, Cl⁻, I⁻, orother halogens.

[0037] The invention of course also encompasses a labeled nucleotideobtainable by a method as disclosed above.

[0038] In addition, the invention encompasses a labeling substance forlabeling nucleotides by a method as disclosed above. The labelingsubstance of the invention has the formula

[0039] wherein X and A have the above meanings and the labeling moietycomprises a label and a spacer as described above. Of course thelabeling substances of the invention can also be used for labelingpurposes other than labeling nucleotides. It was found that numerous(bio-) organic compounds, i.e. nearly every bio-organic molecule whichcontains an accessible sulphur or nitrogen atom, for example proteins,can be labeled with the platinum compounds of the invention.

[0040] A great advantage of the invention arises from the use of theplatinum compounds having formula (I) as linkers in the methods ofpreparing labeled nucleotides according to the invention. These linkerscan be prepared by very convenient and reliable methods.

[0041] In WO92/01699 the starting compounds disclosed for preparinglabeled platinum compounds are platinum(II)(ethylenediamine)dichlorideand platinum(II)(ethylenediamine)(Me₂SO)Cl. The first one can beobtained commercially, the second one (the preferred one) must besynthesized. In the dichloride compound, the Cl-ions are less readilysubstituted by a label or a nucleotide, respectively. In the lattercase, the total nucleotide labeling time will be appreciably longer, upto several hours, instead of several minutes.

[0042] The methods for preparing the linkers that are used in the methodof labeling nucleotides according to the invention are based on theselection of suitable starting compounds of the formula PtE₄ wherein Eis an electronegative group, preferably a halogen or NO₃ ⁻ or SO₃ ⁻. Thereaction, which is described in the examples, of these startingcompounds with e.g. ethylenediamine is very simple and efficient.Moreover, this reaction leads to very suitable symmetric intermediatecompounds for producing labeled nucleotides. A major advantage of usingthese compounds is that when a stabilizing bridge for the resultingplatinum compound has to be attached that no blocking reagents have tobe employed. Another advantage is that the resulting intermediatecompounds can again be labeled without the use of blocking agents.Therefore steps removing blocking agents can be eliminated completely.Furthermore the yields of these reactions are very high. Yet anotheradvantage of the use of these symmetrical starting compounds is that nomixtures of different resulting compounds can be formed, which mayinterfere with the following reaction and reduce yield or require extraseparation steps.

[0043] A very suitable intermediate compound according to the inventionis platinum(II)(ethylenediamine)(NO₃)₂. This substance can very easilybe provided with a suitable spacer and a labeling group, resulting inlabeling substances which can, through substitution of the remainingNO₃-group be linked to a nucleotide quite easily. Furthermore themethods for producing these compounds and the resulting compounds do notinvolve highly toxic substances such as DMSO.

[0044] The intermediate compounds can be labeled with any suitable label(also known as marker) through a spacer as disclosed hereinabove.

[0045] Furthermore, the known advantages (from WO92/01699 for instance)are of course also obtained with the present methods and compounds.Another advantage of the platinum compounds is that they can be detectedmore or less directly by using the platinum as a nucleus for depositingsilver or other metal crystals.

[0046] By binding the labeling substance to a nucleotide residue, DNA orRNA molecules, be it single stranded or otherwise, can be easilydetected, but it also allows for the production of probes forhybridization techniques wherein unlabeled DNA/RNA molecules hybridizeto the labeled probe. The platinum linker labeled nucleotides do hardlyinterfere with the hybridization, if at all. Also, this techniqueobviates the use of modified nucleotides in preparing probes.

[0047] Nucleotides modified in accordance with the practices of thisinvention and oligo- and polynucleotides into which the modifiednucleotides have been incorporated or oligo- and polynucleotides thathave been directly modified using these novel platinum compounds may beused as probes in biomedical research, clinical diagnostics andrecombinant DNA technology.

[0048] Other advantages and embodiments of the invention will becomeclear from the following experimental part and the examples.

EXPERIMENTAL

[0049] Synthesis of Intermediate Platinum Compounds

[0050] These compounds, i.e. the linkers having formula (I), may beprepared by a process which involves:

[0051] (a) reacting a compound having the structure:

[0052]  with KI in a suitable solvent under suitable conditions so as toform a iodated platinum compound having the structure:

[0053] (b) reacting said iodated platinum compound with ethylenediaminein a suitable solvent so as to form a diethyleneamine iodated platinumcompound and represented by the formula Pt(en)I₂ and having thestructure:

[0054] (c) reacting said compound with AgNO₃, the reaction being carriedout in a suitable solvent, under suitable conditions so as to form acompound having the structure:

[0055] (d) reacting said compound with KCl in a suitable solvent undersuitable conditions so as to form a compound having the structure:

[0056] (e) reacting said compound with AgNO₃ in a suitable solvent,under suitable conditions so as to form a compound having the structure:

[0057] (f) recovering said compound as modified platinum startingcompound for the synthesis of hapten-bound Pt(en) compounds for use asDNA and/or RNA label.

Example 1

[0058] A. Preparation of Pt(en)-diamine Starting Material.

[0059] Preparation of Platinumethylenediamine(NO₃)₂: Starting Material.

[0060] Pt(en)(NO₃)₂

[0061] All Reactions are Performed in the Dark.

[0062] Dissolve 1 gram potassium tetrachloroplatinate (II), K₂PtCl₄ (2.4mmol, Sigma) in 50 ml millipore (filtered water) and stir at roomtemperature. Add 10 equivalents of potassium iodide, KI (24 mmol, 3.999g, Sigma). The colour of the solution will immediately turn from orangeinto dark red (K₂PtI₄), stir for 5 minutes.

[0063] Add one equivalent ethylenediamine (2.4 mmol, 160.8743 μl, Merck11=0.9 kg) after diluting 161 μl ethylenediamine in 5 ml millipore veryslowly to the platinum solution, mix this X solution for 1 hour at roomtemperature.

[0064] A yellow/brown precipitate, Pt(en)I₂, will be formed and theliquid standing above should be clear.

[0065] Filter the solution through a 1.0 μm membrane filter(Schleicher&Schuell), wash the precipitate with millipore, ethanol anddiether (in this order). Dry the Pt(en)I₂ for at least 4 hours in avacuum dryoven at 37° C.

[0066] Weigh the dried Pt(en)I₂ (˜1.07 g) and suspend it in 45 mlmillipore/5 ml aceton, the solution will be cloudy. Add 1.95 equivalentof AgNO₃ (M=169.9, Sigma). Stir the reaction overnight at roomtemperature.

[0067] Filter the solution through a 1.0 μm membrane filter, theprecipitate is Silveriodide, AgI, the filtrate should be clear.

[0068] Add to 0.5 ml of the filtrate, Pt(en)(NO₃)₂, an excess of KCl orNaCl and make sure that no white precipitate is formed immediately afteradding the excess of KCl or NaCl. If no white precipitate (only a yellowone) is formed than add an excess of KCl or NaCl to the entire filtrate.After the yellow precipitate is formed filter the solution and wash theprecipitate (Pt(en)Cl₂) with millipore, ethanol and diether.

[0069] Dry the precipitate for at least 4 hours in a vacuum dryoven at37° C.

[0070] Weigh the dry Pt(en)Cl₂ (M=326,1), and suspend it in 45 mlmillipore/5 ml aceton and stir the cloudy suspension. Add 1.95equivalent AgNO₃ and stir the solution overnight at room temperature.The colour of the solution will become white, due to the formation ofAgCl.

[0071] Filter the solution in the dark and evaporate the filtrate toremove the aceton by rotation evaporation untill 25 ml of the filtrateis left. The filtrate is then freezedried. The product is checked by NMRor Infrared Absorption Spectroscopy.

[0072] B. Preparation of Pt(tmen)-diamine Starting Material.

[0073] Preparation ofPlatinum-N,N,N′N′-tetramethylethylenediamine(NO₃)₂: Starting Material.

[0074] Pt(tmen)(NO₃)₂

[0075] All Reactions are Performed in the Dark

[0076] Repeat the entire procedure of Example 1A, but useN,N,N′,N′-tetramethylethylenediamine instead of ethylenediamine.

EXAMPLE 2

[0077] A. Preparation of [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃)

[0078] Dissolve Pt(en)(NO₃)₂ (18.2 mg, 0.048 mmol) in 10 ml of Milliporewater and heat until dissolving. Dissolve BioDadoo (20 mg, 0.053 mmol,purchased from Boehringer Mannheim) in 5 ml of Millipore water. Add thetwo solutions together and adjust the pH to 8 by 0.1 N NaOH, react forat least 3 hours at 50° C. Isolate the end product by freeze drying.

[0079] B. Preparation of [Pt(tmen)(BioDadoo-NH₂)(NO₃)](NO₃)

[0080] Dissolve Pt(tmen)(NO₃)₂ (35 mg, 0.08 mmol) in 12.5 ml ofMillipore water and heat until dissolving. Dissolve BioDadoo (32 mg,0.085 mmol) in 10 ml of Millipore water. Add the two solutions togetherand adjust the pH by 0.1 N NaOH, react for at least 4 hours at 50° C.Isolate the end product by freeze drying.

[0081] C. Preparation of [Pt(en)(DigDadoo-NH₂)(NO₃)](NO₃)

[0082] Dissolve Pt(en)(NO₃)₂ (5 mg, 0.013 mmol) in 5 ml of Milliporewater and heat until dissolving. Dissolve DigDadoo (9 mg, 0.016 mmol,purchased from Boehringer Mannheim) in 5 ml of Millipore water. Add thetwo solutions together and react for at least 4 hours at 50° C. Isolatethe endproduct by freeze drying.

EXAMPLE 3

[0083] A. Preparation of a Labeled dGTP

[0084] Dissolve [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃) (9 mg, 0.012 mmol) in 2ml of Millipore water. Add 2′-deoxyguanosine-5′-triphosphate (2.3 mg,0.004 mmol) and adjust the pH to 6. Incubate for 24 hours at 50° C.,freeze-dry and redissolve in Millipore water (1 ml) and filter through amembrane filter. Apply the mixture to a FPLC with MonoQ and purify witha linear gradient from 100% Millipore water to 100% 1M NH₄HCO₃, collectand pool appropriate fraction and isolate by freeze drying. Dissolve theproduct in a 100 mM solution of triethylamine ammonium acetate (TEAA) (1ml) and apply to a Reversed Phase HPLC (C18) with a linear gradient from100% 100 mM TEAA to 50% 100 mM TEAA/50% 100 mM TEA/acetonitrile (l/lv/v), collect and pool the appropriate fraction and remove solvents byrepeated evaporation in vacuo Pass the product overa cation exchanger(Dowex) in the lithium form, isolate the product by freeze drying.

[0085] B. Preparation of a Labeled 5-AA-dUTP

[0086] Dissolve [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃) (6 mg, 0.008 mmol) in 2ml of Millipore water. Add 2′-deoxyuridine-5-aminoallyl-5′-triphosphate(2 mg, 0.004 mmol) and adjust the pH to 8. Incubate for 24 hours at 50°C., freeze-dry and redissolve in Millipore water (1 ml) and filterthrough a membrane filter. Apply the mixture to a FPLC with MonoQ andpurify with a linear gradient from 100% Millipore water to 100% 1MNH₄HCO₃, collect and pool appropriate fraction and isolate by freezedrying. Dissolve the product in a 100 mM solution of triethylamineammonium acetate (TEAA) (1 ml) and apply to a Reversed Phase HPLC (C18)with a linear gradient from 100% 100 EM TEAA to 50% 100 mM TEAA/50% 100mM TEAA/acetonitrile (l/l v/v), collect and pool the appropriatefraction and remove solvents by repeated evaporation in vacuo. Pass theproduct overa cation exchanger (Dowex) in the lithium form, isolate theproduct by freeze drying.

EXAMPLE 4

[0087] Reaction For Coupling Pt(en)-compounds to DNA

[0088] Typical Reaction For Labeling DNA Molecules with a Pt-CompoundAccording to the Invention.

[0089] 5 μg of double stranded DNA is sonicated or DNase treated toyield fragments of 300-500 bp. 6 μg of Pt(en)-compound is added and thevolume is adjusted to 50 μl with demineralised water. The reactionmixture is incubated at 65° C. for 1 hour. Non-bound Pt(en)-compound isblocked by adding 100 μl of a NADDTC solution. The Pt(en)-compoundlabeled DNA is purified on a sphadex G-50 column. Readily labeled andpurified DNA is stored at −20° C. or used directly in a DNA probe basedassay. Pt(en)-compound labeled DNA probes can be stored at least 2 yearsat −20° C. without loss of activity and/or specificity. All applicationsmentioned are carried out with probes labeled according to thisprotocol.

EXAMPLE 5

[0090] Biotin Labeling of DNA Probes with[Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃) (BioDadoo-ULS).

[0091] Introduction

[0092] The labeling method has been used to label DNA probes with Biotinfor In Situ Hybridization (ISH). In this example labeling proceduresincluding the protocols and data for quality control procedures arepresented. For Biotin labeling a plasmid cloned total DNA of HumanPapilloma Virus type 6 (HPV-6, 40% GC basepairs) was used.

[0093] Experimental Procedures

[0094] Plasmid DNA Preparation

[0095] Total DNA of Human Papilloma Virus type 6 was cloned into vectorpSp-64. Plasmid DNA was transferred into E.coli (C-600) and plated ontoampicillin containing LB plates Single colonies were grown overnight inlarge culture. Plasmid DNA was isolated according to the method ofBirnboim and Doly¹, purified by Sepharose C1-2B columnchromatography(Pharmacia) and checked for inserts by restriction-enzyme-analyses.Plasmid DNA concentration was determined by A260/280 absorbtion. Afterethanol precipitation the DNA was reconstituted in 10 mM TRIS/HCl pH7.2, 0.3 mM EDTA to a final concentration of 1 μg/μl (batch#150894).Subsequently this DNA was sonicated (Soniprep 150., MSE) for 3 times 10minutes (amplitude 5 microns) on ice.

[0096] The size of the resulting DNA fragments was determined by 2%agarose gel electrophoresis and found to be in between 200-400 basepairs(batch#051094).

[0097] Plasmid DNA Labeling and Purification

[0098] Plasmid HPV-6 DNA was labeled with BioDadoo-ULS by mixing thefollowing reagents: plasmid HPV-6 DNA (batch# 051094)  5 μl (1 μg/μl)BioDadoo-ULS labeling reagent  8 μl (1 μg/μl) (batch# BX940830)Demineralised water (<0.2/μS/cm) 37 μl

[0099] The 50 μl reaction mixture was incubated for 15 minutes at 85° C.

[0100] Excess of labeling reagent was captured by adding 50 μl sodiumdiethyldithiocarbamate (2% solution in demineralised water) andincubating for 30 minutes at room temperature. Unbound BioDadoo-ULS wasremoved, using a S300 HR microspin column (Pharmacia), by size exclusionchromatography. Eluent volume was adapted to 500 μl giving a 10 ng/μlbiotin HPV-6 probe concentration (batch#061094).

[0101] Quality Control For Detection Limits

[0102] The detection limit of the biotin probe of the invention wasdetermined by direct spot blot and reversed filterhybridizationaccording to the following protocols:

[0103] Direct Spot Blot

[0104] HPV-6 probe (batch#061094) labeled with biotin according to theinvention was 10-fold serially diluted into spot buffer comprising 900mM sodium chloride, 90 mM sodium citrate and 200 μg/ml single strandedsalmon sperm DNA giving a dilution series varying from 1000-0.1 pgbiotin probe per μl. 1 μl spots were applied onto nitrocellulosemembrane and incubated for 2 hours at 80° C. to bind the DNA. The biotinprobe was visualized using a streptavidin-alkaline phosphatase conjugate(Sigma) combined with. a NBT/BCIP precipitating substrate solution(Sigma) according to the following protocol:

[0105] Membranes were soaked in TBS solution containing 0.5% tween20(TBST) for 5 minutes.

[0106] Membranes were incubated with Strep-AP (3 DEA U/ml) in TBST for15 minutes at 37° C.

[0107] NC-membranes were washed 3 times 5 minutes in TBS solutionfollowed by a 5 minute wash step in demineralised water.

[0108] Membranes were incubated with NBT/BCIP substrate solution for 15minutes at 37° C., subsequently washed in demineralised water and airdried.

[0109] Results

[0110] By using this method the detection limit of the biotin DNA probeaccording to the invention was found to be less than 1 pg.

[0111] Reversed Filterhybridization

[0112] HPV-6 DNA (batch#051094) was1 in 10 diluted in 0.1N NaOH,incubated at 100° C. for 5 minutes and directly placed on ice for 5minutes to make DNA single stranded.

[0113] A 10-fold serial dilution was made in cold 0.1N NaOH to give aseries varying from 10,000-1 pg DNA per μl. 1 μl spots were applied ontoNylon membrane (Boehringer Mannheim) and air dried.

[0114] HPV-6 DNA probe that was labeled with biotin according to theinvention was diluted in 5× SSPE 0.5% SDS solution to yield aconcentration of 200 ng/ml.

[0115] This sloution was incubated for 5 minutes at 100° C. and placeddirectly on ice for 5 minutes.

[0116] Nylon membranes containing target DNA were soaked in 2× SSC for 5minutes and subsequently incubated with the single stranded probesolution for 2 hours at 37° C.

[0117] Excess of the biotin probe was removed by three chances in 2×SSPE 0.1% SDS for 10 minutes at 37° C. followed by a 5 minutes TBSTincubation.

[0118] The biotin probe of the invention was visualized by performingthe same protocol as described in the direct spot blot method.

[0119] Results

[0120] By using this procedure the detection limit of the biotin probeaccording to the invention was found to be less than 10 pg

[0121] Performance in in situ Hybridization

[0122] The test material consisted of 6 μm paraffin sections of a HPV-6posotive cervical condyloma mounted on organosilane coated glass slides.

[0123] The following protocol was applied (unless otherwise stated stepsare at room temperature):

[0124] 1 Paraffin sections were dewaxed in 3 changes of xylene andhydrated in graded ethanol.

[0125] 2 Sections were rinsed in TBST for 5 minutes.

[0126] 3 Sections were digested in 0.25% pepsin in 0.1N HCl for 30minutes at 37° C., dehydrated in graded ethanol and air dried.

[0127] 4 10 μl of probe solution was applied to a section and coveredwith a coverslip.

[0128]  Probe solution consisted of biotin HPV-6 probe DNA labeledaccording to the invention (batch#061094) in a concentration of 2 ng/μldissolved in hybridization mixture comprising 0.6M NaCl, 0.06M sodiumcitrate, 35% formamid, 10% dextransulphate, 2,5× Denhardts and 10 μg/mlsingle stranded salmon sperm DNA.

[0129] 5 Slides were placed on a hot plate set at 95° C. for 5 minutesto denature probe and target DNA simultaneously.

[0130] 6 Hybridization was performed by placing the slides in ahumidified chamber at 37° C. for 2 hours.

[0131] 7 Coverslips were removed and slides were washed in 3 changes of15 mM NaCl, 1.5 mM sodium citrate and 5% formamid for 10 minutes at 37°C.

[0132] 8 Slides were rinsed in TBST.

[0133] 9 Sections were incubated with Streptavidin AP copnjugate(3DEAU/ml in TBST) for 15 minutes at 37° C.

[0134] 10 Slides were washed in TBST (3×) and demineralised water (1×)for 5 minutes.

[0135] 11 Sections were incubated with NBT/BCIP substrated solution for15 minutes at 37° C.

[0136] 12 Slides were washed in demineralised water (3×) for 1 minuteand sections were mounted in glycerol/gelatin.

[0137] Results

[0138] By using the sections showed blue/purple precipitates at thesites of HPV-6 infected cells and minor background in the remainingtissue.

[0139] Conclusions

[0140] The results demonstrate that DNA labeled according to theinvention has good detection limits. The present method is very wellsuited for research, routine and for industrial production of labelednucleic acids, as the method is fast and easy to perform, verysensitive, and does not include any enzymaic step, which makes it highlyreproducible and fitted for an overall low cost production. The methodof the invention offers a useful alternative equaling conventionalnon-isotopic labeling methods.

GENERAL REFERENCES

[0141] 1. Maniatis T., Sambrook J., Fritsch E. F., Molecular Cloning,Second Edition, Cold Spring Harbor Laboratory Press, ISBN 0-8769-309-6.

[0142] 2. Keller G. H., Manak M. M., DNA probes, Stockton Press, ISBN0-333-47659-X.

[0143] Applications

[0144] 1. The Use of Pt-DNA Linkers of the Invention in the so CalledLIDIA Technique: Linked DNA Immuno Assay.

[0145] The LIDIA technique enables the quantitative analysis of smallamounts of DNA (or RNA) e.g. after a PCR amplification of the startingmaterial. The technique is sensitive and specific, due to the use ofspecific DNA(RNA) probes in accordance with the invention and easy toperform, because of the quick DNA(RNA) Pt-labeling steps of theinvention.

[0146] Description of the Technique:

[0147] The technique uses fast Pt labeling compounds of the invention tolabel DNA(RNA) probes.

[0148] This technique is possible with 3 different approaches.

[0149] 1. Linking DNA probes molecules to a surface by using a Ptcompound in accordance with the invention which cross-link DNA moleculesirreversibly to plastic, nylon or nitrocellulose. Detection of DNAtargets can then be accomplished by using classically labeled DNA/RNAprobes.(nick translation or chemical modification, random priming)

[0150] 2. Linking a detectable group to the DNA, to render a DNAmolecule into a so-called DNA probe. Binding of DNA compounds to asurface can then be accomplished by using classic techniques known toscience (covalent linking to specially treated microtiter plates, bakingof DNA molecules onto nitrocellulose or binding of DNA molecules tonylon membranes.

[0151] 3. A combination of techniques 1 and 2

[0152] Approach 1

[0153] An immobilized DNA probe can be used to catch specific targetmolecules in a sample by using a hybridization technique. Detection offormed hybrids can be done by using different techniques, e.g. a secondlabeled DNA probe can be used to hybridize with a different site on thetarget DNA molecule to form a sandwich hybrid. The label can hen bedetected by using state of the art immunological detection and colouringtechniques.

[0154] Approach 2

[0155] A volume containing (amplified) detectable DNA(RNA) is directlylabeled according to the protocol in accordance with the invention.

[0156] Excess label is quenched by adding NaDDTC or Thioureum. Thisapproach distinguishes itself from other techniques by the fact that thetarget molecule is labeled in contrast to other assay were labeledDNA(RNA) probes are used to detect the target. The quick bindingcapacity of the Pt-label compound of the invention enables a DNA bindingstep as a routine step in a diagnostic test procedure (normal bindingtimes are 60 minutes at 65° C.).

[0157] A second step is performed in a microtiter plate precoated with atarget specific probe. Incubation is allowed to the formation of stable“Labelled target” and probe hybrids. The direct labeling of targetmolecules enables the omission of laborious double hybridizationtechniques where one probe is used to catch the target and anotherlabeled probe is used to detect the immobilized target.

[0158] In this method the probes are covalently linked to the microtiterplate to the surface of the wells. The second incubation step has thecharacter of a liquid hybridisation and therefore can be performed veryrapidly. This is one of the main innovative features of this approach toquantitive DNA hybridisation techniques.

[0159] Approach 3

[0160] Both for the immobilization of DNA probes or DNA targets and forthe labeling of DNA probes and targets the newly developed Pt system canbe used. These two DNA linking techniques can be combined into one assaywhere both the “catcher” and the “detector” are linked to a secondsubstance (either a detectable group like biotin, digoxigenin or acarrier surface like a plastic stick, microtiter plate or a membrane).

[0161] Examples of the Technique: the Detection of STD RelatedMicroorganisms in Human Diagnostics (Chlamydia, Syfillis, AIDS, Herpes,Gonorrhoea, Hep. B,)

[0162] 2. The Use of Pt-DNA Labels of the Invention in Combination withTest Strip Procedures and Formats. The “DNA Dipstick”.

[0163] The DNA dipstick technique enables the qualitative andsemi-quantitative analysis of small amounts of DNA(or RNA) e.g. after aPCR amplification or freely present in samples of body fluids (blood,urine, sweat etc.) The technique is sensitive and specific, due to theuse of specific DNA(RNA) probes and easy to perform because of the quickDNA(RNA) Pt-labeling steps according to the invention.

[0164] The universal labeling characteristics of the newly developed Ptlabel can be used in 3 ways to achieve a bound DNA(RNA) molecule.

[0165] 1. It can be used to attach a detectable marker group to apolynucleotide sequence.

[0166] 2. It can be used to attach polynucleotide sequences irreversiblyto a solid phase (plastic, membranes, latex beads, hydrosols ormicrotiter plate wells).

[0167] 3. A combination of 1 and 2

[0168] ad 1:

[0169] In this example there is a twofold approach to the detection ofbiolytes biological analytes in test samples. Firstly a DNA probe can belabeled with the newly developed Pt labeling compound. This labeledprobe can then be used to detect preformed hybrids on a membrane formedbetween the target DNA sequence and a primary probe. It is essential inthis method that the primary probe recognizes a different sequence onthe target than the secondary Pt labeled probe. In practice, this can beachieved for instance with RNA hybridization were a POLY A probe is usedas a primary probe to immobilize all RNA (recognizable by its polyTtails) to a membrane.

[0170] The second approach differs slightly in that in this case thetarget can be labeled in the test sample fluid, because of the fast andvery specific Pt labeling characteristics. A procedure like this wouldcomprise a catch of the labeled target with an immobilized specificunlabeled DNA probe on a suitable membrane. Hence a dipstick version forDNA/RNA applications.

[0171] ad 2:

[0172] To immobilize DNA probes or target DNA, a non-labeled Pt compound(that is a Pt compound with 2 free binding sites) can be used to act asa bridge between DNA and the surface of carriers (plastic, membrane,microtiter plates etc.)

[0173] It greatly enhances the usability of DNA sequences as catchermolecules in diagnostic assays, since there are little substances knownto science that bind readily DNA in a spontaneous way. Introducing thisPt bridge molecule a wide field of new applications for the DNAtechnology has come go within reach.

[0174] ad 3: a Combination of Example 1 and 2

[0175] General: the use of the Pt compound of the invention in latex orhydrosol assays is particularly interesting. The compound enables thecoupling of DNA molecules to small particles. The DNA molecules can behybridized to target material. A positive reaction is visualized by anagglutination of the particles, due to crosslinking of the DNA hybridparticle compounds.

[0176] A test like this can be made quantative, the rate ofagglutination can be tuned and measured at a specific wavelength.Especially gold particles have the intrinsic characteristic that a shiftin optimal wavelength occurs after agglutination.

[0177] 3. Detection of Platinated DNA Probes of the Invention with thethe Silver-Enhancement Technique.

[0178] Platinated DNA/RNA probe can be employed in hybridisation methodsto detect DNA/RNA sequences in sample material. The introduction of aplatinum compound at the site of the target enables the deposition of Agmolecules in a chemical reaction especially designed to reduce ionicsilver to metallic silver. At the site of a Pt nucleus a decompositionof metallic silver(black) occurs due to the catalytic effect of the Ptnucleus.

[0179] Ionic silver is reduced by a reducing agent (e.g. Na-borohydrid,Hydrochinon) in solution. In a constant ratio the amount of silverdeposited on the Pt is proportional to the length of the enhancementincubation. Visualisation of a non-visible Pt nucleus can beaccomplished by the empirical observation of the appearace of a blackspot in the test sample.

[0180] The black spots indicate the site of specific probes binding andthus the site of specific target location. The technique enables a quickand easy diagnostic procedure for the detection of variousmicroorganisms and gene translocations/abnormalities.

1. A method for labeling nucleotides comprising the steps of: reacting areactive moiety of a linker of the formula

 wherein X represents any stabilizing bridge and wherein A and Brepresent the same or different reactive moieties, with an electrondonating moiety of a spacer, which spacer comprises a chain having atleast four atoms and at least one heteroatom in the chain, which spacerfurther comprises said electron donating moiety at one end of the chainand a reactive moiety at the other end of the chain; reacting thereactive moiety of said spacer with a label; reacting the other reactivemoiety of said linker with a nucleotide.
 2. A method according to claim1 wherein the label is biotin, avidin, streptavidin, digoxygenin or afunctional equivalent thereof.
 3. A method according to claim 1 or 2wherein the nucleotide is adenine, thymidine, cytosine, guanine oruridine or a derivative thereof.
 4. A method according to any of thepreceding claims wherein the electron donating moiety is an amine or athiolate anion.
 5. A method according to claim 4 wherein the amine is anaromatic amine.
 6. A method according to any of the preceding claimswherein at least one heteroatom is an oxygen atom.
 7. A method accordingto any of the preceding claims wherein the spacer comprises no more than20 carbon atoms in the chain and wherein the chain is essentiallynon-branched.
 8. A method according to claim 7 wherein the spacer is1,8-diamino-3,6-dioxaoctane.
 9. A method according to claim 7 whereinthe spacer is an oligolysine or a polylysine.
 10. A method according toclaims 1-4 wherein the linker is reacted with a labeling moietycomprising a label and a spacer, which labeling moiety has the formula

or the formula

wherein X represents any stabilizing bridge, Z and Z′ represent anon-leaving ligand and n is an integer of from 2 to
 10. 11. A methodaccording to claim 10, wherein Z and/or Z′ represent an NH₃, NH₂R, NHR₂,or NR₃ group, wherein R represents an alkyl group having from 1 to 6carbon atoms.
 12. A method according to any of the preceding claims,wherein X represents an aliphatic diamine.
 13. A method according toclaim 12 wherein X represents an aliphatic diamine having 2-6 carbonatoms.
 14. A method according to claim 13 wherein X is an ethylenediamine group.
 15. A method according to claims 12-14 wherein one orboth of the nitrogen atoms of the aliphatic diamine are shielded.
 16. Amethod according to claim 15 wherein one or both of the nitrogen atomsof the aliphatic diamine are substituted with an alkyl group of from 1to 6 carbon atoms.
 17. A method according to claim 16 wherein one orboth of the nitrogen atoms of the aliphatic diamine are substituted withone or two methyl groups.
 18. A method according to any of the precedingclaims wherein A and/or B are selected from the group consisting ofNO₃-, SO₃ ⁻, Cl⁻, I⁻, or other halogens.
 19. A method according to anyof the preceding claims wherein A and B are the same.
 20. A labelednucleotide obtainable by a method according to any of the precedingclaims.
 21. A labeled nucleotide according to claim 20 wherein thespacer is an oligolysine or a polylysine.
 22. A labeling substancehaving the formula

wherein X represents any stabilizing bridge, A represents a reactivemoiety and the labeling moiety comprises a label and a spacer, whichspacer comprises a chain having at least four atoms and at least oneheteroatom in the chain, which spacer further comprises an electrondonating moiety at the end of the spacer distal from the label.